The Nanostructured Self-Assembly and Thermoresponsiveness in Water of Amphiphilic Copolymers Carrying Oligoethylene Glycol and Polysiloxane Side Chains

Amphiphilic copolymer self-assembly is a straightforward approach to obtain responsive micelles, nanoparticles, and vesicles that are particularly attractive for biomedicine, i.e., for the delivery of functional molecules. Here, amphiphilic copolymers of hydrophobic polysiloxane methacrylate and hydrophilic oligo (ethylene glycol) methyl ether methacrylate with different lengths of oxyethylenic side chains were synthesized via controlled RAFT radical polymerization and characterized both thermally and in solution. In particular, the thermoresponsive and self-assembling behavior of the water-soluble copolymers in water was investigated via complementary techniques such as light transmittance, dynamic light scattering (DLS), and small-angle X-ray scattering (SAXS) measurements. All the copolymers synthesized were thermoresponsive, displaying a cloud point temperature (Tcp) strongly dependent on macromolecular parameters such as the length of the oligo(ethylene glycol) side chains and the content of the SiMA counits, as well as the concentration of the copolymer in water, which is consistent with a lower critical solution temperature (LCST)-type behavior. SAXS analysis revealed that the copolymers formed nanostructures in water below Tcp, whose dimension and shape depended on the content of the hydrophobic components in the copolymer. The hydrodynamic diameter (Dh) determined by DLS increased with the amount of SiMA and the associated morphology at higher SiMA contents was found to be pearl-necklace-micelle-like, composed of connected hydrophobic cores. These novel amphiphilic copolymers were able to modulate thermoresponsiveness in water in a wide range of temperatures, including the physiological temperature, as well as the dimension and shape of their nanostructured assemblies, simply by varying their chemical composition and the length of the hydrophilic side chains.


Introduction
Amphiphilic polymers are a class of materials that can be employed to obtain selfassembled responsive nanomaterials with great potential in medicine and physiology [1,2]. Even though block copolymers are generally recognized as the workhorse of self-assembled copolymer nanostructures, in recent years, great attention has been directed to the study of the behavior in this field of amphiphilic homopolymers [3][4][5][6] and linear statistical/random copolymers. These materials have the advantage of being accessible via easy, one-step, synthetic procedures such as the straightforward free-radical polymerization [7,8] of hydrophilic and hydrophobic comonomers, but can also display more sophisticated properties when dispersity, chain lengths, and composition are controlled [9][10][11][12] by means of reversible deactivation radical polymerization techniques such as ATRP, RAFT, etc.
The reaction conditions for the preparation of the other PEGMA-co-SiMAx copolymers are summarized in Table S1.
The purification procedure was different for copolymers with different SiMA contents because of the different solubilities. The crude products PEGMA-co-SiMA10 and PEGMAco-SiMA17 were precipitated three times from dichloromethane solutions into n-hexane; the crude product PEGMA-co-SiMA29 and PEGMA-co-SiMA45 were purified by dialysis of different copolymer solutions (~0.2 g mL −1 ) against water, water/THF (1/1 v/v) and THF in the order, using a dialysis membrane (Repligen (Boston, MA, USA) Spectra/Por Biotech RC, 3.5-5 kD cut-off, 16 mm). After dialysis, the copolymers were recovered from the THF solution by evaporating the solvent under vacuum.
The reaction conditions for the preparation of the other TEGMA-co-SiMAx copolymers are summarized in Table S1.
The purification procedure was different for copolymers with different SiMA contents because of the different solubilities. The crude products of TEGMA-co-SiMA4 and TEGMAco-SiMA6 were precipitated three times from dichloromethane solutions into n-hexane. TEGMA-co-SiMA65 was precipitated three times from dichloromethane solutions into ethanol. TEGMA-co-SiMA15, TEGMA-co-SiMA19 and TEGMA-co-SiMA28 were purified by dialysis of different copolymer solutions (~0.2 g mL −1 ) against water, water/THF (1/1 v/v), and THF in that order, using a dialysis membrane (Repligen (Boston, MA, USA) Spectra/Por Biotech RC, 3.5-5 kD cut-off, 16 mm). After dialysis, the copolymers were recovered from the THF solution by evaporating the solvent under vacuum.

Characterization
1 H measurements were carried out on a Bruker Avance 400 (400 MHz, Billerica, MA, USA) spectrometer with deuterated solvents at room temperature. The sample concentration was approximately 30 g L −1 . Gel permeation chromatography (GPC) was carried out using a Jasco (Hachioji-shi, Tokyo, Japan) PU-2089 Plus liquid chromatograph equipped with two PL gel 5 µm mixed-D columns, a Jasco RI-2031 Plus refractive index detector, and a Jasco (Hachioji-shi, Tokyo, Japan) UV-2077 Plus UV/vis detector. Samples were filtered via a 0.2 µm PTFE filter before injection. The measurements of number and weight average molecular weights and dispersity (M n , M w , Ð), relative to PMMA narrow standards, were obtained from refractive index detector curves, in chloroform as the mobile phase (1 mL min −1 ) at 30 • C, maintained by a Jasco (Hachioji-shi, Tokyo, Japan) CO 2063 Plus column thermostat.
Differential scanning calorimetry (DSC) analysis was performed with a Mettler (Columbus, OH, USA) DSC 922e Module from −130 to 100 • C at heating/cooling rate of 10 • C min −1 under a dry nitrogen flow. The glass transition temperature (T g ) was taken as the inflection temperature in the second heating cycle.
A Shimadzu (Kyoto, Japan) 2450 UV-vis spectrophotometer was used for measuring of transmittance of solutions at a fixed wavelength of 700 nm as a function of temperature, in quartz cuvettes with a 10 mm optical path. The cloud point temperature (T cp ) was defined at the middle of the signal drop. UV-vis spectra (200-800 nm) were acquired using a PerkinElmer (Waltham, MA, USA) Lambda 650 spectrophotometer. Solutions in solvents of different polarities were analyzed in a concentration range of 10 −3 -10 −4 M in quartz cuvettes with an optical path of 10 mm.
A Malvern (Malvern, Worcestershire, UK) Zetasizer Nanoparticle analyzer (detection angle = 173 • ) or a Beckman Coulter (Brea, CA, USA) Delsa Nano C particle analyzer (detection angle = 166 • ) were used for dynamic light scattering (DLS) measurements of the prefiltered copolymer solutions (5 µm PTFE or cellulose acetate filters to reduce contaminant interferences). Intensity, volume, and number distributions from CONTIN analysis in the instrument software were obtained from the raw intensity autocorrelation function of at least five different repetitions. The copolymers were dissolved in HPLC-grade water, chloroform, tetrahydrofuran, and dimethylformamide of (0.2 µm filtered).
Small-angle X-ray scattering (SAXS) was executed via an Anton Paar (Graz, Austria) SAXSess system with a temperature-controlled sample cell with a quartz capillary (1 mm diameter, 10 µm wall thickness), sealed by vacuum-tight screwcaps on both ends. The vacuum during the measurements was ≈ 1 mbar. The solvent background contribution to the scattering intensity was recorded separately and subtracted on a relative scale (after normalization to the same transmissions) from all the samples SAXS intensities (20 g L −1 copolymer solutions to allow adequate scattering intensity for analysis). The Debyeflex 3003 (GE-Inspections technologies, Frankfurt, Germany) X-ray generator source was operated at 40 kV and 50 mA with a sealed-tube Cu anode (Cu-Kα radiation source, λ = 0.154 nm). The X-ray beam was Goebel-mirror-focused and Kratky(line)-slit collimated with a final rectangular shaped (17 mm × 0.25 mm). A 1D MYTHEN-1k microstrip solid-state detector recorded the spectra in transmission mode, with a magnitude of the scattering vector q between 0.01 to 5 nm −1 . Given 2θ as the scattering angle (with respect to the incident beam) and λ as the wavelength of the X-rays, the sample to detector distance of 307 mm corresponded to a total 2θ region of 0.14 • -7 • , applying the conversion q = 4π sin(θ)/λ. The exposure time was typically 60 s times 5, with a waiting time of 10 min between the different temperature steps for all spectra.

Synthesis of PEGMA-co-SiMAx and TEGMA-co-SiMAx Copolymers
The two sets of PEGMA-co-SiMAx and TEGMA-co-SiMAx amphiphilic copolymers contained the same hydrophobic (though not lipophobic) polydimethylsiloxane methacrylate (SiMA, M n = 680 g/mol, number average degree of polymerization n~6) component and hydrophilic poly(ethylene glycol) methyl ether methacrylate (PEGMA, M n = 475 g/mol, number average degree of polymerization n~9) or triethyleneglycol methyl ether methacrylate (TEGMA, n = 3) (Figure 1). component and hydrophilic poly(ethylene glycol) methyl ether methacrylate (PEGMA, Mn = 475 g/mol, number average degree of polymerization n ~ 9) or triethyleneglycol methyl ether methacrylate (TEGMA, n = 3) (Figure 1). For both sets of copolymers, a reversible addition-fragmentation chain-transfer (RAFT) polymerization was chosen which employed 2-cyano-2-propyl benzodithioate as chain transfer agent (CTA) (Figure 1). The aromatic group in this CTA was easily detectable by 1 H NMR, which is useful for the determination of the molecular weight and degree of polymerization of the different copolymer samples. The molar ratio between the two comonomers in the feed mixture was varied over a rather large composition range (5-70 mol% SiMA) ( Table S1).
The RAFT polymerization was carried out with toluene as solvent at 70 °C under nitrogen, using 2,2′-azobis(2-methylpropionitrile) (AIBN) as the thermal initiator with a CTA:AIBN molar ratio of 5:1 for all the copolymerization runs. The reaction products were named PEGMA-co-SiMAx and TEGMA-co-SiMAx, where x, the molar percentage of SiMA, was determined by 1 H NMR analysis, as well as the overall composition of the copolymer ( Figures S1 and S2). The integral areas of the signals at 7.4-8 ppm of the aromatic protons in the CTA, 0.1 ppm for Si(CH3)2 protons of SiMA, 3.4 ppm for OCH3 protons of PEGMA or TEGMA, and 4-4.5 ppm for COOCH2 protons of both repeating units were used to calculate the number average degree of polymerization of the copolymers; the compositions are reported in Table 1.  For both sets of copolymers, a reversible addition-fragmentation chain-transfer (RAFT) polymerization was chosen which employed 2-cyano-2-propyl benzodithioate as chain transfer agent (CTA) (Figure 1). The aromatic group in this CTA was easily detectable by 1 H NMR, which is useful for the determination of the molecular weight and degree of polymerization of the different copolymer samples. The molar ratio between the two comonomers in the feed mixture was varied over a rather large composition range (5-70 mol% SiMA) ( Table S1).
The RAFT polymerization was carried out with toluene as solvent at 70 • C under nitrogen, using 2,2 -azobis(2-methylpropionitrile) (AIBN) as the thermal initiator with a CTA:AIBN molar ratio of 5:1 for all the copolymerization runs. The reaction products were named PEGMA-co-SiMAx and TEGMA-co-SiMAx, where x, the molar percentage of SiMA, was determined by 1 H NMR analysis, as well as the overall composition of the copolymer ( Figures S1 and S2). The integral areas of the signals at 7.4-8 ppm of the aromatic protons in the CTA, 0.1 ppm for Si(CH 3 ) 2 protons of SiMA, 3.4 ppm for OCH 3 protons of PEGMA or TEGMA, and 4-4.5 ppm for COOCH 2 protons of both repeating units were used to calculate the number average degree of polymerization of the copolymers; the compositions are reported in Table 1.  linear up to 8 h of polymerization where the total monomer conversion reached 64%. This implies a first-order kinetics of the consumption of monomers during the polymerization [50]. The plot also showed an induction time of about two hours, consistent with what already reported for polymerization mediated by the dithiobenzoate CTA used in this reaction [51,52]. As expected for a controlled RAFT polymerization, the number average degree of polymerization DP n grew linearly with conversion ( Figure 2b), in agreement with the theoretical values given by Equation (1): where p is the monomer conversion and [M] 0 and [CTA] 0 are the initial molar concentrations of both comonomers and chain transfer agent, respectively.
where p is the monomer conversion and [M]0 and [CTA]0 are the initial molar concentrations of both comonomers and chain transfer agent, respectively. GPC analysis confirmed the occurred copolymerization, and the copolymer curves were monomodal with a generally relatively low dispersity Đ ≤ 1.33, apart from copolymers PEGMA-co-SiMAx with a higher mole content (≥29%) of SiMA. The discrepancies in the Mn determined by GPC and 1 H NMR analysis were due to the calibration of the GPC setup with PMMA standards, that are characterized by a dissimilar hydrodynamic and conformational behavior, compared to the atypical one of amphiphilic copolymers.
In keeping with the precise control of the macromolecular structure achieved by the RAFT copolymerization, the chemical composition of the copolymers was modulated over a predetermined wide molar range of both counits. This in turn enabled the production of several narrowly dispersed copolymers featuring diverse amphiphilic characters for investigation of self-assembling in solutions. While a few examples of amphiphilic copolymers based on fluorinated comonomers are reported in the literature [14,15,42,[53][54][55], very little is known about the thermoresponsiveness and self-assembly in the water of amphiphilic copolymers based on siloxane comonomers [56].  GPC analysis confirmed the occurred copolymerization, and the copolymer curves were monomodal with a generally relatively low dispersity Ð ≤ 1.33, apart from copolymers PEGMA-co-SiMAx with a higher mole content (≥29%) of SiMA. The discrepancies in the M n determined by GPC and 1 H NMR analysis were due to the calibration of the GPC setup with PMMA standards, that are characterized by a dissimilar hydrodynamic and conformational behavior, compared to the atypical one of amphiphilic copolymers.

Differential Scanning Calorimetry
In keeping with the precise control of the macromolecular structure achieved by the RAFT copolymerization, the chemical composition of the copolymers was modulated over a predetermined wide molar range of both counits. This in turn enabled the production of several narrowly dispersed copolymers featuring diverse amphiphilic characters for investigation of self-assembling in solutions. While a few examples of amphiphilic copolymers based on fluorinated comonomers are reported in the literature [14,15,42,[53][54][55], very little is known about the thermoresponsiveness and self-assembly in the water of amphiphilic copolymers based on siloxane comonomers [56].

Differential Scanning Calorimetry
Thermal characterization of synthesized copolymers was performed by differential scanning calorimetry (DSC) analyses (heating/cooling rates 10 • C min −1 ) with the objectives of identifying the glass transition temperature (T g ) and its specific heat capacity change (∆C p ) as well as the melting temperature (T m ) with its enthalpy variation (∆H m ). All samples were examined between −130 and 100 • C. Copolymers PEGMA-co-SiMAx were semicrystalline, whereas copolymers TEGMA-co-SiMAx were completely amorphous ( Figure 3). For both copolymer systems, the overall thermal behavior depended on chemical composition. Thermal characterization of synthesized copolymers was performed by differential scanning calorimetry (DSC) analyses (heating/cooling rates 10 °C min −1 ) with the objectives of identifying the glass transition temperature (Tg) and its specific heat capacity change (ΔCp) as well as the melting temperature (Tm) with its enthalpy variation (ΔHm). All samples were examined between −130 and 100 °C. Copolymers PEGMA-co-SiMAx were semicrystalline, whereas copolymers TEGMA-co-SiMAx were completely amorphous ( Figure 3). For both copolymer systems, the overall thermal behavior depended on chemical composition. The temperatures of glass and melting transitions and the associated specific heat and enthalpy changes are reported in Table 2. For copolymers with low amounts of SiMA, only one glass transition was detected similar to those of the homopolymers, p(PEGMA) (Tg = −63 °C) and p(TEGMA) (Tg = −48 °C). The copolymers with higher SiMA contents (≥45 mol% and 15 mol% for PEGMA-co-SiMAx and TEGMA-co-SiMAx, respectively) presented a second glass transition in the range −124 °C ÷ −115 °C that is similar to the behavior of the p(SiMA) homopolymer (Tg = −107 °C). This result suggests that the hydrophilic and the hydrophobic components were chemically incompatible and underwent microphase separation in the bulk. Moreover, a melting transition was also detected for PEGMA-co-SiMAx associated with the crystalline regions of PEGMA counits. Such a transition did not occur for TEGMA-co-SiMAx copolymers, given the shorter length of the oxyethylenic side chains, which is consistent with the completely amorphous nature of the corresponding homopolymer. The melting temperature was found to decrease from −9 °C to −18 °C as the content of PEGMA in the copolymer decreased from 90 to 55 mol% with a reduction in the melting enthalpy. The decrease in the crystallinity degree was due to the incorporation of SiMA counits as defective elements. Table 2. Thermal properties of the copolymers PEGMA-co-SiMAx and TEGMA-co-SiMAx.

Copolymer
Tg (a) (°C) The temperatures of glass and melting transitions and the associated specific heat and enthalpy changes are reported in Table 2. For copolymers with low amounts of SiMA, only one glass transition was detected similar to those of the homopolymers, p(PEGMA) (T g = −63 • C) and p(TEGMA) (T g = −48 • C). The copolymers with higher SiMA contents (≥45 mol% and 15 mol% for PEGMA-co-SiMAx and TEGMA-co-SiMAx, respectively) presented a second glass transition in the range −124 • C ÷ −115 • C that is similar to the behavior of the p(SiMA) homopolymer (T g = −107 • C). This result suggests that the hydrophilic and the hydrophobic components were chemically incompatible and underwent microphase separation in the bulk. Moreover, a melting transition was also detected for PEGMA-co-SiMAx associated with the crystalline regions of PEGMA counits. Such a transition did not occur for TEGMA-co-SiMAx copolymers, given the shorter length of the oxyethylenic side chains, which is consistent with the completely amorphous nature of the corresponding homopolymer. The melting temperature was found to decrease from −9 • C to −18 • C as the content of PEGMA in the copolymer decreased from 90 to 55 mol% with a reduction in the melting enthalpy. The decrease in the crystallinity degree was due to the incorporation of SiMA counits as defective elements. Table 2. Thermal properties of the copolymers PEGMA-co-SiMAx and TEGMA-co-SiMAx. 3.3. Self-Assembly in Solution 3.

Light Transmittance Measurements
Transmittance at a wavelength of λ = 700 nm of PEGMA-co-SiMAx and TEGMA-co-SiMAx solution in water was measured using a UV-vis spectrophotometer, thermostated (±0.1 • C) at different temperatures. Copolymers TEGMA-co-SiMAx with a SiMA content greater than 19 mol% were poorly soluble or completely insoluble in water and could not be investigated. The light transmittance values of solutions of PEGMA-co-SiMAx (with x = 10, 17, and 29 mol%) in water (10 g L −1 ) were plotted in Figure 4a,b as a function of temperature in a heating and cooling cycle, respectively.

Light Transmittance Measurements
Transmittance at a wavelength of λ = 700 nm of PEGMA-co-SiMAx and TEGMA-co-SiMAx solution in water was measured using a UV-vis spectrophotometer, thermostated (±0.1 °C) at different temperatures. Copolymers TEGMA-co-SiMAx with a SiMA content greater than 19 mol% were poorly soluble or completely insoluble in water and could not be investigated. The light transmittance values of solutions of PEGMA-co-SiMAx (with x = 10, 17, and 29 mol%) in water (10 g L −1 ) were plotted in Figure 4a,b as a function of temperature in a heating and cooling cycle, respectively.
The curves in Figure 4a show a sharp reduction in the transmittance values from about 100% to 0% on heating to a critical temperature, named the cloud point temperature (Tcp), at which copolymer aggregation into large multichain aggregates occurred. This phenomenon was also easily visually detected, since at room temperature the polymeric solution was transparent and homogeneous, whereas it turned into a cloudy dispersion at and above Tcp. A cooling ramp was also carried out to evaluate the effective reversibility of the transition process (Figure 4b). For the solutions of copolymers with a SiMA content lower than 17 mol%, the transition was reversible with negligible hysteresis, whereas for the other samples, there was a slight thermal hysteresis by about 6-8 °C.  The Tcp was found to strongly depend on the type of hydrophilic component and its content in the copolymer. In particular, for both sets of copolymers, Tcp increased as the content of the hydrophilic component increased, tending to the values of the respective homopolymers pPEGMA (Tcp = 90 °C) and pTEGMA (Tcp = 47 °C) [59]. This result confirms that the amphiphilic copolymers of this work showed a similar thermoresponsive behavior as their respective homopolymers, by which hydrogen bonds between water and hydrophilic oxyethylene side chains are broken at Tcp and intermacromolecular polar The curves in Figure 4a show a sharp reduction in the transmittance values from about 100% to 0% on heating to a critical temperature, named the cloud point temperature (T cp ), at which copolymer aggregation into large multichain aggregates occurred. This phenomenon was also easily visually detected, since at room temperature the polymeric solution was transparent and homogeneous, whereas it turned into a cloudy dispersion at and above T cp . A cooling ramp was also carried out to evaluate the effective reversibility of the transition process (Figure 4b). For the solutions of copolymers with a SiMA content lower than 17 mol%, the transition was reversible with negligible hysteresis, whereas for the other samples, there was a slight thermal hysteresis by about 6-8 • C.
The T cp was found to strongly depend on the type of hydrophilic component and its content in the copolymer. In particular, for both sets of copolymers, T cp increased as the content of the hydrophilic component increased, tending to the values of the respective homopolymers pPEGMA (T cp = 90 • C) and pTEGMA (T cp = 47 • C) [59]. This result confirms that the amphiphilic copolymers of this work showed a similar thermoresponsive behavior as their respective homopolymers, by which hydrogen bonds between water and hydrophilic oxyethylene side chains are broken at T cp and intermacromolecular polar bonds among copolymer chains are formed, thereby giving rise to multichain aggregates. Moreover, for a similar content of the hydrophilic component in the copolymer, the T cp decreased by more than 50 • C (copolymer concentration 10 g L −1 ) by passing from PEGMA to TEGMA counits (Table 3). According to the literature [4,59], the longer oxyethylene side chains guarantee a higher hydrophilicity and assist water solubility of the non-aggregated (co)polymer chains in the aqueous medium over an extended temperature interval. Table 3. Cloud temperature of copolymer solutions in water at different concentrations (error on T cp within ± 0.2 • C).

Copolymer
Concentration PEGMA-co-SiMA10 To investigate the influence of the concentration on the thermoresponsive behavior, aqueous solutions of selected copolymers were prepared at various concentrations and analyzed through transmittance measurements by varying the temperature (Table 3). By increasing the concentration of the copolymer, a reduction in the value of T cp was observed down to a plateau, consistent with a lower critical solution temperature (LCST)-type behavior (Figure 4c,d).

Dynamic Light Scattering
Dynamic light scattering (DLS) measurements were carried out on the copolymers PEGMA-co-SiMAx and TEGMA-co-SiMAx to investigate the ability of these amphiphilic copolymers to form self-assembled nanostructures in water. Furthermore, the influence of temperature on these systems was assessed. For each PEGMA-co-SiMAx water-soluble sample, aqueous solutions with a concentration of 10 g L −1 of copolymer were analyzed by DLS varying the temperature in the range 25-90 • C. On the other hand, copolymers TEGMA-co-SiMAx were analyzed at a reduced concentration of 1 g L −1 to minimize the turbidity of the solution. Copolymers with a SiMA content higher than 19 mol% were not analyzed because of their complete insolubility. The hydrodynamic diameter (D h ) values at room temperature and at a temperature above T cp are collected in Table 4 for the two series of copolymers. Table 4. Average hydrodynamic diameters (D h ) by DLS of copolymer solutions in various organic solvents (20 g L −1 ) and water (10 g L −1 ) at 25 • C and above T cp for PEGMA-co-SiMAx and TEGMAco-SiMAx.
The DLS measurements of the PEGMA90-co-SiMA10 and TEGMA-co-SiMA6 copolymers will be described as a typical example (Figures 5a and 5b, respectively). A drastic sharp increase in D h at a temperature above T cp was observed, confirming the light transmittance results (Figure 4). In fact, the cloud point observed could be related to an abrupt change in the aggregation state of the amphiphilic copolymers in water. This finding was attributed to the formation of smaller micellar nanostructures at T < T cp and larger aggregates at T ≥ T cp . The D h values measured in a cooling cycle were similar to those obtained during heating, confirming the reversibility of the thermoresponsive phenomenon.
For any copolymer solution investigated herein (Table 4), we observed that increasing the SiMA mole content in the copolymer resulted in an increase in the size of the nanostructures observed at T < T cp , and a decrease in the D h measured at T > T cp up to the point reached for the copolymer PEGMA-co-SiMA45, for which the difference between the two values was the smallest one (30 nm).
The effect of copolymer concentration on self-assembly was also tested. DLS measurements were carried out by varying the concentration (from 0.5 to 30 g L −1 ) of copolymer in the water at 25 • C and at T > T cp . The case of PEGMA-co-SiMA29 will be discussed in detail as an example (Figure 5c). These analyses showed that at a temperature below T cp , the D h of the nanostructures did not change significantly with the increase in concentration of the copolymer in the water solution, with a behavior that is compatible with a micellar, close aggregation mechanism, for the concentration range investigated. Differently, at a temperature above T cp , the micellar nanoparticles collapse into larger aggregates that showed an open aggregation mechanism, with an increase in the size of the aggregate at higher copolymer concentrations (Figure 5c,d).
copolymer in the water at 25 °C and at T > Tcp. The case of PEGMA-co-SiMA29 will be discussed in detail as an example (Figure 5c). These analyses showed that at a temperature below Tcp, the Dh of the nanostructures did not change significantly with the increase in concentration of the copolymer in the water solution, with a behavior that is compatible with a micellar, close aggregation mechanism, for the concentration range investigated. Differently, at a temperature above Tcp, the micellar nanoparticles collapse into larger aggregates that showed an open aggregation mechanism, with an increase in the size of the aggregate at higher copolymer concentrations (Figure 5c,d). The effect of organic solvents on the self-assembly of the amphiphilic copolymers at room temperature was also investigated. Chloroform, tetrahydrofuran (THF) and dimethylformamide (DMF) were used as solvents with lower selectivity than water towards the hydrophilic/hydrophobic component of the copolymers. Measured average hydrodynamic diameters are reported in Table 4. As an example, in Figure 6a, the intensity size distribution was also compared with that in water for copolymer PEGMA-co-SiMA10. Particle size in organic solvents was generally found to be 5 nm ≤ D h ≤ 15 nm, that was significantly smaller than in water. The hydrodynamic diameter of the nanostructures in aqueous solution increased with increasing SiMA content in the copolymer, going from 17 nm to 71 nm for the PEGMA-co-SiMA series and from 13 nm to 37 nm for the TEGMA-co-SiMA series. The relatively large sizes of the nanostructures formed in aqueous solutions at T < T cp seem to be compatible with the formation of multichain micellar nanoparticles rather than single-chain, unimer micelles that are normally characterized by D h < 10 nm [15,60]. In agreement with the results from SAXS analysis (see below Section 3.3. 3) we assume that in the tested organic solvents the copolymers were totally solvated, assuming a typical random coil conformation. In contrast, in aqueous solution, the polymer chains self-assembled into micellar nanoparticles due to the favorable interactions of water with oxyethylenic side chains and hydrophobic interaction among the siloxane ones. This assembly resulted in distinct hydrophobic compartments whose non-polar nature was confirmed by the solvatochromic shift of CTA residue absorption in UV-vis spectra (see Section 3.3.4 below).
towards the hydrophilic/hydrophobic component of the copolymers. Measured hydrodynamic diameters are reported in Table 4. As an example, in Figure 6a, the size distribution was also compared with that in water for copolymer PEGMA-co Particle size in organic solvents was generally found to be 5 nm ≤ Dh ≤ 15 nm, significantly smaller than in water. The hydrodynamic diameter of the nanostru aqueous solution increased with increasing SiMA content in the copolymer, go 17 nm to 71 nm for the PEGMA-co-SiMA series and from 13 nm to 37 nm for the co-SiMA series. The relatively large sizes of the nanostructures formed in solutions at T < Tcp seem to be compatible with the formation of multichain nanoparticles rather than single-chain, unimer micelles that are normally charact Dh < 10 nm [15,60]. In agreement with the results from SAXS analysis (see below 3.3. 3) we assume that in the tested organic solvents the copolymers were totally assuming a typical random coil conformation. In contrast, in aqueous solu polymer chains self-assembled into micellar nanoparticles due to the interactions of water with oxyethylenic side chains and hydrophobic interactio the siloxane ones. This assembly resulted in distinct hydrophobic compartmen non-polar nature was confirmed by the solvatochromic shift of CTA residue abso UV-vis spectra (see Section 3.3.4 below).

Small-Angle X-ray Scattering
To investigate the morphology of the micellar nanoparticles in solution, so synthesized amphiphilic copolymers were analyzed by small-angle X-ray s (SAXS) measurements. In particular, aqueous and THF solutions (20 g L −1 ) of cop PEGMA-co-SiMA10, PEGMA-co-SiMA45, TEGMA-co-SiMA6 and TEGMA-co were analyzed by SAXS. In Figure 7a we report the scattering intensity I(q) of al in water as a function of the scattering vector q after background subtraction.
The Guinier plots (Figure 7b), linear in the region of low q values (1/Rg < q were used to determine the radius of gyration Rg (Table 5)

Small-Angle X-ray Scattering
To investigate the morphology of the micellar nanoparticles in solution, some of the synthesized amphiphilic copolymers were analyzed by small-angle X-ray scattering (SAXS) measurements. In particular, aqueous and THF solutions (20 g L −1 ) of copolymers PEGMAco-SiMA10, PEGMA-co-SiMA45, TEGMA-co-SiMA6 and TEGMA-co-SiMA15 were analyzed by SAXS. In Figure 7a we report the scattering intensity I(q) of all samples in water as a function of the scattering vector q after background subtraction.
The Guinier plots (Figure 7b), linear in the region of low q values (1/R g < q < 1.3/R g ), were used to determine the radius of gyration R g (Table 5) of the particles. In the water solution, the Guinier curves showed a positive variation with respect to linearity at low q values (q 2 < 0.4 nm -2 ). This deviation suggests the presence of attractive interactions between the molecules in aqueous solution, confirming the formation of multichain micellar nanoparticles. Additionally, R g values calculated for all the samples in water were higher than those in THF, in agreement with what was shown by the DLS measurements. The combined SAXS and DLS results suggest that these copolymers self-assembled in water solution in small aggregates due to the intermacromolecular interactions among copolymer chains.
values (q 2 < 0.4 nm -2 ). This deviation suggests the presence of attractive interactions between the molecules in aqueous solution, confirming the formation of multichain micellar nanoparticles. Additionally, Rg values calculated for all the samples in water were higher than those in THF, in agreement with what was shown by the DLS measurements. The combined SAXS and DLS results suggest that these copolymers self-assembled in water solution in small aggregates due to the intermacromolecular interactions among copolymer chains.  Kratky plots (q 2 I(q) vs. q) of copolymer solutions in water (selective solvent) and THF (non-selective solvent) are compared in Figure 8. TEGMA-co-SiMA6 and TEGMA-co-SiMA15 displayed in THF (Figure 8b) ascending q 2 I(q) curves until a plateau was reached in the high q region, typical of polymers that adopt a random coil conformation. By contrast, Kratky plots of the copolymer in water (Figure 8a) showed a small peak followed by a large shoulder at higher q. This shape of the plot indicates the partial degree of folding within the nanostructures, due to the hydrophobic interactions among the hydrophobic portions of the copolymer such as the siloxane chains. Moreover, the real space p(r)function was obtained via indirect Fourier transformation [61] of the scattering curve I(q) in reciprocal space. This kind of plot is also called distance distribution function and helps to elucidate the shape and conformation of polymer particles [62,63].  Kratky plots (q 2 I(q) vs. q) of copolymer solutions in water (selective solvent) and THF (non-selective solvent) are compared in Figure 8. TEGMA-co-SiMA6 and TEGMA-co-SiMA15 displayed in THF (Figure 8b) ascending q 2 I(q) curves until a plateau was reached in the high q region, typical of polymers that adopt a random coil conformation. By contrast, Kratky plots of the copolymer in water (Figure 8a) showed a small peak followed by a large shoulder at higher q. This shape of the plot indicates the partial degree of folding within the nanostructures, due to the hydrophobic interactions among the hydrophobic portions of the copolymer such as the siloxane chains. Moreover, the real space p(r)-function was obtained via indirect Fourier transformation [61] of the scattering curve I(q) in reciprocal space. This kind of plot is also called distance distribution function and helps to elucidate the shape and conformation of polymer particles [62,63]. Figure 9 demonstrates that the maximum dimension of the copolymer particle in water solution was in any case on the order 10-15 nm. Moreover, for PEGMA-co-SiMA10 and TEGMA-co-SiMA6 the maximum of the p(r)-function correlated well with the R g values obtained from the Guinier analysis. However, the asymmetric profile of the curve as well as the presence of several humps (Figure 9a,b) were indicative of an anisotropic shape of the non-homogeneous and non-compact aggregates [64]. On the other hand, the p(r)-function plot for the copolymers richer in SiMA (PEGMA-co-SiMA45 and TEGMA-co-SiMA15) (Figure 9c,d) more clearly indicated the presence of multicore, pearl necklace micelles in solution. For these samples, a first peak (r ≈ 2 nm) correlates to the diameter of one globule slightly smaller than the size of a single polymer chain in THF (R g ≈ 2−2.3 nm); a second peak (r ≈ 8-10 nm) arises from the pairing distances between neighboring globules [65,66]. There are also larger structures present which could not be resolved well with our SAXS measurements as they were beyond our resolution and which are the reason for the sharp cutoff of the p(r)-functions at r > 10 and r > 12 nm, respectively, in Figure 9b Figure 9 demonstrates that the maximum dimension of the copolymer particle in water solution was in any case on the order 10-15 nm. Moreover, for PEGMA-co-SiMA10 and TEGMA-co-SiMA6 the maximum of the p(r)-function correlated well with the Rg values obtained from the Guinier analysis. However, the asymmetric profile of the curve as well as the presence of several humps (Figure 9a,b) were indicative of an anisotropic shape of the non-homogeneous and non-compact aggregates [64]. On the other hand, the p(r)-function plot for the copolymers richer in SiMA (PEGMA-co-SiMA45 and TEGMAco-SiMA15) (Figure 9c,d) more clearly indicated the presence of multicore, pearl necklace micelles in solution. For these samples, a first peak (r ≈ 2 nm) correlates to the diameter of one globule slightly smaller than the size of a single polymer chain in THF (Rg ≈ 2−2.3 nm); a second peak (r ≈ 8-10 nm) arises from the pairing distances between neighboring globules [65,66]. There are also larger structures present which could not be resolved well with our SAXS measurements as they were beyond our resolution and which are the reason for the sharp cutoff of the p(r)-functions at r > 10 and r > 12 nm, respectively, in Figure 9b-d.
Thus, the pearl necklace structure (Figure 9e) seemed to acquire greater dimensional uniformity and conformational homogeneity with the increase in SiMA content in the copolymer. Increasing the percentage of such a component could lead to the formation of more compact, folded domains within the single polymer chain arising from the stronger hydrophobic interactions of SiMA side chains. Such folded domains are connected by unfolded flexible portions. Differently, copolymers richer in PEGMA, being overall more soluble in water were less susceptible to fold intramolecularly in compact nanostructures to shield the hydrophobic compartment, thus aggregating structurally less-defined nanoparticles. Necklace-like micelle morphology was recently reported in the literature to describe the aggregation in water of amphiphilic copolymers of PEG-acrylamide and dodecyl acrylamide [23]. It is also reported for other systems such as copolymers of sodium maleate and dodecyl vinyl ether [7,[67][68][69]. The UV-vis spectrum of the RAFT chain transfer agent used is characterized by a maximum of absorption at λmax = 303 nm and a second minor peak at λ = 521 nm in CHCl3 as shown in Figure 10a. Moreover, λmax showed solvatochromic behavior, with a bathochromic shift of ~10 nm from 298 nm to 308 nm with increasing solvent polarity from Figure 9. Distance distribution p(r)-function of water solutions (20 g L −1 ) of copolymers PEGMAco-SiMAx and TEGMA-co-SiMAx (a-d). Schematic representation of the self-assembly in "pearl necklace" micelles of the copolymers with higher SiMA content (e). Thus, the pearl necklace structure (Figure 9e) seemed to acquire greater dimensional uniformity and conformational homogeneity with the increase in SiMA content in the copolymer. Increasing the percentage of such a component could lead to the formation of more compact, folded domains within the single polymer chain arising from the stronger hydrophobic interactions of SiMA side chains. Such folded domains are connected by unfolded flexible portions. Differently, copolymers richer in PEGMA, being overall more soluble in water were less susceptible to fold intramolecularly in compact nanostructures to shield the hydrophobic compartment, thus aggregating structurally less-defined nanoparticles. Necklace-like micelle morphology was recently reported in the literature to describe the aggregation in water of amphiphilic copolymers of PEG-acrylamide and dodecyl acrylamide [23]. It is also reported for other systems such as copolymers of sodium maleate and dodecyl vinyl ether [7,[67][68][69].

Solvatochromism of the RAFT CTA
The UV-vis spectrum of the RAFT chain transfer agent used is characterized by a maximum of absorption at λ max = 303 nm and a second minor peak at λ = 521 nm in CHCl 3 as shown in Figure 10a. Moreover, λ max showed solvatochromic behavior, with a bathochromic shift of~10 nm from 298 nm to 308 nm with increasing solvent polarity from n-hexane/PDMS to water (Table 6).  Thus, the residue of the CTA attached to the macromolecular chain end is also anticipated to be sensitive to the nano-environment derived from the self-assembly of the copolymers in water. In fact, the CTA residue in pPEGMA showed a λmax in water solutions similar to those in water itself (307 nm), but a decrease in λmax was detected for water solutions of the amphiphilic copolymers. In particular, λmax was found to decrease by increasing the SiMA content in the copolymer, reaching a minimum value of 299 nm for PEGMA-co-SiMA45 (Figure 10b, Table 6). This finding demonstrates that the CTA  Thus, the residue of the CTA attached to the macromolecular chain end is also anticipated to be sensitive to the nano-environment derived from the self-assembly of the copolymers in water. In fact, the CTA residue in pPEGMA showed a λ max in water solutions similar to those in water itself (307 nm), but a decrease in λ max was detected for water solutions of the amphiphilic copolymers. In particular, λ max was found to decrease by increasing the SiMA content in the copolymer, reaching a minimum value of 299 nm for PEGMA-co-SiMA45 (Figure 10b, Table 6). This finding demonstrates that the CTA residue is exposed to an environment characterized by a polarity comparable to that of n-hexane and PDMS. This is consistent with the preferential formation of more compact folded domains with hydrophobic siloxane core shielded by hydrophilic PEG chains (Figure 9e). This was also showed by SAXS analysis by copolymer with higher SiMA contents.

Conclusions
PEGMA (or TEGMA)-co-SiMAx amphiphilic copolymers with modulated solubility in water, were synthesized by RAFT controlled polymerization. All water-soluble copolymers demonstrated reversible LCST-like thermoresponsive features, typically with a cloud-point temperature T cp which was predominantly dependent on the hydrophilic oligo(ethylene glycol) side chain length and the percentage of the hydrophobic SiMA comonomer. By easily varying these structural parameters, the T cp of copolymer water solutions (10 g L -1 ) was tailored to span a wide range (from~85 • C to~27 • C), passing across the physiological temperature. Thermoresponsiveness was also affected by the concentration of the copolymer in solution.
The copolymer nanostructures in water below T cp were deeply characterized by DLS and SAXS. The D h increased with the SiMA content, varying from 17 nm to 70 nm in going from PEGMA-co-SiMA10 to PEGMA-co-SiMA45. These dimensions suggested the formation of multichain micellar nanostructures whose multicore pearl necklace-like morphology was clearly proved by SAXS for both PEGMA and TEGMA-based copolymers richer in SiMA counits. By contrast, copolymers showed a random coil conformation in THF, given the non-selective nature of this solvent as opposite to water. The covalently linked RAFT CTA moiety exhibited a solvatochromic effect as a result of its preferential compartmentalization in the apolar environment typical of the hydrophobic cores of the micelles in water.
In conclusion, these amphiphilic copolymers, owing to simple chemical modification, were susceptible to modulations in their nano-assembling capacity and responses to external stimuli. In particular, the copolymers of the series TEGMA-co-SiMAx, with the shorter hydrophilic side chain and characterized by the lower T cp range (41-27 • C), are the most promising for application as thermoresponsive carriers with a trigger across the human physiological temperature. In several examples, the dimension of such nano-assemblies was in the sub-100 nm realm. These features make them worth investigating as smart carriers for the encapsulation and delivery of hydrophobic molecules, especially drugs and bioimaging agents. A gradual increase in the hydrophobic character of the micelle compartments was observed by increasing the SiMA content in the copolymer; thus, we can speculate that different micelles acting as carriers could accommodate functional molecules with different degrees of hydrophobicity.